CN102634581A - Fluorescent quantitative PCR (Polymerase Chain Reaction) qualitative detection method for yak-derived fiber composition - Google Patents
Fluorescent quantitative PCR (Polymerase Chain Reaction) qualitative detection method for yak-derived fiber composition Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) qualitative detection method for yak-derived fiber composition. The fluorescent quantitative PCR qualitative detection method comprises the following steps of: performing PCR amplification with the DNA (Deoxyribonucleic Acid) of yakwool as a template, and detecting fluorescent signals of amplification products, wherein a reaction system for the PCR amplification contains a specific primer pair for amplification of yak-derived fiber composition and specific probes for the yak-derived fiber composition. The fluorescent quantitative PCR qualitative detection method can be used for easily and quickly detecting and identifying whether the yak-derived fiber composition exists in textiles or not; and due to the good specificity and excellent sensitivity of the designed specific primer pairs and specific probes, the method can be used for detecting yakwool with proportion being 1%(wt).
Description
Technical field
The invention belongs to bionic technical field, specifically, is yak source property fibre composition quantitative fluorescent PCR qualitative checking method.
Background technology
Yak is a kind of Mammals that can under the severe environment of highland and severe cold, survive, and also is the unique member that the weaving utility value is arranged of bovid.Yak is divided into Bos mutus and tame yak, and Bos mutus cries wild ox again, formal name used at school Bos mutus (Po ě phagrt mutrs), and English name wild yak hides name transliteration Asia and returns.Yak is grown in the extremely frigid zones of 3000 meters～5000m of height above sea level, and the severe cold that ability is subzero 30 ℃～40 ℃ is being assembled fine and closely woven fine hair under the thick long batt of yak, these fine hair be yak under high and cold weather conditions, survive provide maybe.More than 1,400 ten thousand of the existing yaks in the whole world mostly are distributed in China Qinghai-Tibet Platean and the above extremely frigid zones of height above sea level 3000m on every side thereof.China is the maximum country of yak number in the world, accounts for 90% of whole world sum.
Yakwool is very similar, as shown in Figure 1 on appearance with purple cashmere, and Fig. 1 is the purple cashmere (left side) of microscopically and the photo of yakwool (right side).All there is pigment group in the both at microscopically, has brought difficulty for discriminating work.Owing to itself have pigment, purple goat and yakwool can be made into dark fabric usually, and this has increased difficulty just further for detection discriminating work.Some lawless persons utilize this similarity, use yakwool as purple cashmere and lead into a trap to the human consumer, get some advantage from it.
The existing standard GB/T 16988-1997 " mensuration of special animal fiber and sheep's wool mixture content " of China is to the description of yakwool: yakwool fiber scale subtended angle is less, and scale density is 100-130/mm, and hair color is black, brown.And under general opticmicroscope, scale is not very clear, detects to get up to waste time and energy.The electron microscopy cost is high, and the relatively more tired rope of sample preparation.
Summary of the invention
The present invention is a kind of qualitative identification method of the yakwool fiber based on DNA detection technology, overcome under the existing optical microphotograph microscopy that scale is unintelligible, the shortcoming that wastes time and energy and; And it is high to have overcome Electronic Speculum inspection cost, and the loaded down with trivial details deficiency of sample preparation.
The objective of the invention is to, a kind of yak source property fibre composition quantitative fluorescent PCR qualitative checking method is provided.
Another object of the present invention is to, the detection kit of a kind of yak source property fibre composition is provided, and the purposes of test kit.
A purpose more of the present invention is, the primer and the probe of a kind of yak source property fibre composition quantitative fluorescent PCR is provided.
The technical problem that will solve required for the present invention, can realize through following technical scheme:
As first aspect of the present invention, a kind of yak source property fibre composition quantitative fluorescent PCR qualitative checking method may further comprise the steps:
The fluorescent signal of step 2, detection amplified production;
It is characterized in that, the reaction system that is used for pcr amplification contain amplification yak source property fibre composition Auele Specific Primer to yak source property fibre composition specific probe.
Wherein, property fibre composition Auele Specific Primer right sequence in said amplification yak source is shown in SEQ ID NO:1 and SEQ ID NO:2.
Wherein, the nucleotide sequence of said yak source property fibre composition specific probe is shown in SEQ ID NO:3.
Wherein, said method also comprises step: in the process of polymerase chain reaction or afterwards, detect the fluorescent signal that yak source property fibre composition specific probe sends.
Wherein, the condition of said pcr amplification is following:
Reaction system: ABI Taqman universal PCR master mix (2 *) 10 μ l, dna profiling 4 μ l, primer 0.6 μ l, probe 2.5 μ l, ddH
2O 4.9 μ l;
Reaction conditions: 95 ℃, 5min; 95 ℃, 15s, 60 ℃, 45s, 40 circulations.
The concentration of primer is 0.3 μ M, and the concentration of probe is 0.25 μ M.
As second aspect of the present invention, the detection kit of a kind of yak source property fibre composition, contain following reagent:
(a) amplification yak source property fibre composition Auele Specific Primer is right;
(b) yak source property fibre composition specific probe.
Further, said test kit also comprises:
(c) marker.
Wherein, said marker is yak source property fibre composition DNA, or contains the segmental DNA of yak source property fibre composition amplification purpose, or yakwool.
Wherein, the right sequence of said Auele Specific Primer is shown in SEQ ID NO:1 and SEQ ID NO:2.
Wherein, the nucleotide sequence of said specific probe is shown in SEQ ID NO:3.
Wherein, the fluorescent signal that sends of said yak source property fibre composition specific probe.
As the third aspect of the invention, the application of the detection kit of a kind of yak source property fibre composition is used for detecting whether there is yak source property fibre composition at textiles.
As fourth aspect of the present invention, the Auele Specific Primer and the specific probe of a kind of yak as claimed in claim 1 source property fibre composition quantitative fluorescent PCR is characterized in that:
The sequence of said Auele Specific Primer is shown in SEQ ID NO:1 and SEQ ID NO:2;
The sequence of said specific probe is shown in SEQ ID NO:3.
As the 5th aspect of the present invention; The Auele Specific Primer of a kind of yak source property fibre composition quantitative fluorescent PCR and the purposes of specific probe, said Auele Specific Primer and specific probe are used for preparing the test kit whether the detection textiles exists yak source property fibre composition.
Beneficial effect of the present invention:
1, can detect easy, fast and identify whether there is yak source property fibre composition in the textiles.
2, the present invention is based on the sequence of yak plastosome 12SrRNA gene, the specificity of design specific primers and specific probe is good.
3, detection method sensitivity is good, can detect the yakwool that proportioning is 1% (wt).
Description of drawings
Fig. 1: the purple cashmere (left side) of microscopically and yakwool (right side).
Fig. 2: reaction system optimization result.
Fig. 3: the blast result of yak primer probe sequence and goat sequence.
Fig. 4: the blast result of yak primer probe sequence and sheep sequence.
Fig. 5: the real-time fluorescence PCR result of yak composition.
Fig. 6: the detection case behind 10 times of gradient dilutions.
Fig. 7: the linear relationship of 10 times of gradient dilutions.
Fig. 8: the yakwool detection case of different proportionings.
Fig. 9: the doubtful cashmere sample that contains yakwool.
Figure 10: the detected result of sample A.
Figure 11: the detected result of sample B.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; The condition that conditioned disjunction manufacturer described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) provides is carried out.
Related " % " in following examples except that specifying, all is weight percentage.
1, the primer and the probe design of the amplification of yak composition real-time fluorescence PCR
Relatively the sequence of goat, sheep, yak plastosome 12SrRNA gene is chosen suitable sequence and is carried out design of primers.Yak source property fibre composition primer amplified product size is 65bp.
The sequence of Auele Specific Primer and probe is following:
YakF：5’-AAGCATCTACACCCCAGTGAGAAT-3’(SEQ?ID?NO：1)
YakR：5’-GCTTGATGCCAGCTCCTCTT-3’(SEQ?ID?NO：2)
Pyak：FAM-CCCTCTAGGTTGTTAAAAT-MGB(SEQ?ID?NO：3)
Designed yak, goat, the total universal primer of sheep simultaneously, universal primer amplified production size is 88bp, and the sequence of universal primer and probe is following:
GsyF：5’-GGGTTTACGACCTCGATGTTG-3’(SEQ?ID?NO：4)
GsyR：5’-ACGTAGGACTTTAATCGTTGAACAAA-3’(SEQ?ID?NO：5)
Pgsy：FAM-ACCGCTATCAAAGGTT-MGB(SEQ?ID?NO：6)
Wherein, FAM representes the fluorescence report group, and MGB representes quenching group.The present invention adopts the fluorescent probe method, and it detects principle is to utilize the fluorescent mark specific probe to come recognition template.Compare with SYBR dye method in the prior art, the specificity of fluorescent mark specific probe of the present invention is stronger.
2, the optimization of the reaction system of yak composition
Get yakwool 5mg and use Progema hair extraction agent box extracting DNA, 50 μ l elutriant eluted dnas are got 4 μ l sample detection.
Reaction system: remove ABI Taqman universal PCR master mix (2 *) 10 μ l, beyond the dna profiling 4 μ l, other compositions are as shown in table 1.
The concentration of primer is 0.3 μ M, and the concentration of probe is 0.25 μ M.
The condition of reaction is: 95 ℃, and 5min; 95 ℃, 15s, 60 ℃, 45s, 40 circulations.
The consumption of other compositions in table 1, the optimizing reaction system
| System | Primer | Probe | ddH 2O |
| 1 | 0.4 | 1 | 6.6 |
| 2 | 0.4 | 2 | 5.6 |
| 3 | 0.6 | 1 | 6.4 |
| 4 | 0.6 | 2 | 5.4 |
| 5 | 0.6 | 2.5 | 4.9 |
Optimum result is as shown in Figure 2.Take all factors into consideration fluorescence signal intensity, factors such as Ct value are selected the real-time fluorescence PCR reaction system of system 5 for amplification yak composition; Be reaction system 5 (composition consumption ABITaqman universal PCR master mix (2 *) 10 μ l, dna profiling 4 μ l, primer 0.6 μ l; Probe 2.5 μ l, ddH
2O 4.9 μ l).
3, the primer of yak composition real-time fluorescence PCR amplification and probe specificity comparison and checking
3.1, the primer and the probe specificity comparison of yak composition real-time fluorescence PCR amplification
Plastosome 12SrRNA of yak and the similarity of goat are 89%, with the similarity of sheep be 87%.The comparison situation of primer and probe and corresponding position such as Fig. 3 and Fig. 4:
The blast result of yak primer probe sequence and goat sequence sees Fig. 3.
The blast result of yak primer probe sequence and sheep sequence sees Fig. 4.
3.2, the primer and the probe specificity experimental verification of yak composition real-time fluorescence PCR amplification
Get yakwool, cashmere, continuous mountain hair 5mg respectively, with progema hair extraction agent box extracting DNA, 70 μ l elutriant eluted dnas.The concentration of primer is 10 μ M, and the concentration of probe is 2 μ M.Respectively with ddH
2O, yakwool, cashmere, continuous mountain hair DNA are that template is carried out the fluorescent PCR amplification.Annealing temperature is 60 ℃ of 45s.Reaction system is following, referring to table 2:
Table 2, yak primer probe specificity confirmatory experiment reaction system
The real-time fluorescence PCR result of yak composition sees Fig. 5.
4, sensitivity detects
With optimizing the mensuration that good reaction system is carried out sensitivity.10 times of gradient dilutions of DNA with the extracting gained can detect 10
4The yak DNA that doubly dilutes.To be prone to simultaneously obscure, the indistinguishable yakwool of microscopy and purple cashmere mix with different proportionings, and wherein the content of yakwool (weight percent) is respectively 100%, 10%, 1%, and it is 1% yakwool that this method of experiment proof can detect proportioning.Result such as Fig. 6-Fig. 8, Fig. 6 are 10 times of detection case behind the gradient dilution, and Fig. 7 is the linear relationship of 10 times of gradient dilutions, and Fig. 8 is the yakwool detection case of different proportionings.
Fig. 7 shows that on weight percent levels, the linear relationship between Ct value and the template copy number is good, can detect yak source property fibre composition DNA concentration.
5, the detection of material object
Have in two doubtful cashmere samples that contain yakwool.
The A sample is the two-layer hair of a dark-grey light gray woolen material, and it is 100% cashmere product that the client applies for checking and examination.Detect through microscopy, A appearance contains cashmere 86.8%, yakwool 9.0%, wool 4.2%.
The B sample contains cashmere 78.1%, yakwool 19.1%, wool 2.8%.The apparent See Figure 9 of sample.
Respectively get A appearance, B appearance one fritter, shred mixing, accurately weighing 5mg uses Progema test kit extracting DNA.Two parallel appearance of each sample.Because of color sample darker, eluted dna repeatedly.Get 4 μ l sample detection, make Gsy simultaneously and detect, as positive control.
Shown in Figure 10 and table 3, the detected value of the Yak of A appearance is respectively 25.55,24.97, and it is 22.65 and 22.11 that the Gsy of the DNA of respective amount detects.
Shown in Figure 11 and table 4, the detected value of black appearance B is all bigger than normal.Black appearance B possibly receive the influence of dyestuff.Certainly sample A contains yakwool, infers that sample B also contains yakwool.
The detected result of table 3, sample A
The detected result of table 4, sample B
According to above enlightenment of the present invention, those skilled in the art will readily understand, can utilize (a) amplification yak source property fibre composition Auele Specific Primer right; (b) yak source property fibre composition specific probe is processed the detection kit of yak source property fibre composition.
Further, said test kit can also comprise (c) marker; Said marker can be yak source property fibre composition DNA, or contains the segmental DNA of yak source property fibre composition amplification purpose, or yakwool.
Obtained (a) amplification yak source property fibre composition Auele Specific Primer to the basis of (b) yak source property fibre composition specific probe on, utilize routine techniques means in the art just can process detection kit, repeat no more here.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (10)
1. yak source property fibre composition quantitative fluorescent PCR qualitative checking method may further comprise the steps:
Step 1, be template, carry out pcr amplification with the DNA of yakwool;
The fluorescent signal of step 2, detection amplified production;
It is characterized in that, the reaction system that is used for pcr amplification contain amplification yak source property fibre composition Auele Specific Primer to yak source property fibre composition specific probe.
2. method according to claim 1 is characterized in that, property fibre composition Auele Specific Primer right sequence in said amplification yak source is shown in SEQ ID NO:1 and SEQ ID NO:2.
3. method according to claim 1 is characterized in that, the nucleotide sequence of said yak source property fibre composition specific probe is shown in SEQ ID NO:3.
4. the method for claim 1 is characterized in that, the condition of said pcr amplification is following:
Reaction system: ABI Taqman universal PCR master mix (2 *) 10 μ l, dna profiling 4 μ l, primer 0.6 μ l, probe 2.5 μ l, ddH
2O 4.9 μ l;
Reaction conditions: 95 ℃, 5min; 95 ℃, 15s, 60 ℃, 45s, 40 circulations.
5. the detection kit of a yak source property fibre composition, contain following reagent:
(a) amplification yak source property fibre composition Auele Specific Primer is right;
(b) yak source property fibre composition specific probe.
6. test kit according to claim 5 is characterized in that, said test kit also comprises:
(c) marker.
7. test kit as claimed in claim 5 is characterized in that, the right sequence of said Auele Specific Primer is shown in SEQ ID NO:1 and SEQ ID NO:2.
8. test kit as claimed in claim 5 is characterized in that, the nucleotide sequence of said specific probe is shown in SEQ ID NO:3.
9. like the application of each described test kit in the claim 5～8, it is characterized in that be used for detecting whether there is yak source property fibre composition at textiles.
10. the Auele Specific Primer and the specific probe of a yak as claimed in claim 1 source property fibre composition quantitative fluorescent PCR is characterized in that,
The sequence of said Auele Specific Primer is shown in SEQ ID NO:1 and SEQ ID NO:2;
The sequence of said specific probe is shown in SEQ ID NO:3.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520938A (en) * | 2016-11-03 | 2017-03-22 | 中国农业大学 | Molecular identification system for distinguishing animal hair shaft fiber species sources |
| CN111044346A (en) * | 2019-12-27 | 2020-04-21 | 武汉纺织大学 | Method for efficiently identifying animal hair and velvet by utilizing heat treatment and microscope |
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100577814C (en) * | 2002-08-27 | 2010-01-06 | 财团法人日本纺织检查协会 | Method of appraising mixing ratio of animal hair fibers in animal hair fiber product |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100577814C (en) * | 2002-08-27 | 2010-01-06 | 财团法人日本纺织检查协会 | Method of appraising mixing ratio of animal hair fibers in animal hair fiber product |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520938A (en) * | 2016-11-03 | 2017-03-22 | 中国农业大学 | Molecular identification system for distinguishing animal hair shaft fiber species sources |
| CN111044346A (en) * | 2019-12-27 | 2020-04-21 | 武汉纺织大学 | Method for efficiently identifying animal hair and velvet by utilizing heat treatment and microscope |
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
| CN113088531B (en) * | 2021-03-25 | 2023-10-17 | 四川省药品检验研究院(四川省医疗器械检测中心) | Bovine-derived ingredient quantitative analysis standard plasmid, preparation and detection methods and application |
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